COMBINED POSITIVE NEGATIVE SELECTION FOR HIGHLY EFFECTIVE PURGING OF PBPC GRAFTS - TOWARDS CLINICAL-APPLICATION IN PATIENTS WITH B-CLL/

Autologous PBPC transplantation is a potentially curative treatment fo r patients with chronic lymphocytic leukemia (CLL), As the autografts are frequently contaminated with large numbers of tumor cells, we have studied double purging of PBPC using immunomagnetic CD34(+) cell sele ction (Isolex 300i) followed by negative depletion with anti-CD19/20/2 3/37-labeled immunomagnetic beads, In four small-scale experiments usi ng PBPC from patients with CLL or leukemic low-grade Lymphoma, double purging resulted in CD34(+) enrichment from 0.9-4.4% to 95.8-99.4%, Ly mphoma cells were always undetectable by FACS and PCR (CDR3 or t(14;18 )) after negative depletion, Next, seven heavily contaminated full gra fts from five patients with CLL or lymphoplasmocytoid immunocytoma wer e subjected to the double purging procedure, resulting in a CD34(+) en richment from 1.6% (0.7-5.6) to 98.5% (96-99.8), The CD34(+) yield aft er double purging was 1.3-6.3 x 10(6)/kg, according to a median recove ry of 32%, The overall reduction of lymphoma cells was 5.6 (>4.6-6) lo g, Although CLL cells were completely absent after purging in five cas es as assessed by FAGS, all grafts remained PCR positive, The first tw o patients have been reinfused with double selected products after mye loablative radiochemotherapy and showed prompt and uneventful hematopo ietic engraftment, We conclude that without significant loss of CD34() cells, negative depletion adds 2 log of CLL cell depletion to CD34() selection, resulting in an overall purging efficacy of more than 5 l og, This combination of positive and negative selection can be success fully applied even to heavily contaminated PBPC grafts.